Abstract. Bone marrow is one of the most radiation-sensitive tissues, and patients acutely exposed to total body irradiation (TBI) doses > 2 Gy develop severe neutropenia, thrombocytopenia, anemia, and lymphopenia within days to weeks of exposure, often dying from infections (due to a lack of neutrophils) and uncontrolled bleeding (insufficient platelets) (referred to as the hematopoietic acute radiation syndrome, or H-ARS). The few drugs that have received FDA approval to treat H-ARS increase survival when administered within 24h of radiation exposure, but do not increase survival when administered at later times such as 48h post-irradiation in animal H-ARS models. In a radiological/nuclear emergency, hundreds of thousands of people potentially will be exposed to > 2 Gy radiation and it is extremely unlikely that they all can be treated within the first 24 hours of radiation exposure. Thus, there is a critical unmet medical need for drugs capable of increasing survival from H-ARS when administered 48h or later post-irradiation. In a pilot study we identified a novel combination of 3 long-acting hematopoietic growth factor proteins that significantly improves 30-day survival of LD40/30 irradiated mice when administered once 48h post-TBI. This is the only drug/drug combination we are aware of capable of increasing survival when dosing is delayed until 48h post-TBI. When dosed once 24h post-TBI, the 3 proteins interact positively with each other to increase survival more than the individual proteins, most evident at high TBI doses (> 9 Gy). By combining the 3 proteins with a 4th radiation mitigator, an angiotensin converting enzyme inhibitor (ACEI), it is possible to obtain 100% 30-d survival of LD95/30 TBI mice, an unprecedented survival rate. This SBIR grant will build upon these exciting findings by (1) determining if the 3 protein combination (+/- the ACEI) can increase survival of mice exposed to higher LD70/30 and LD90/30 radiation doses when the proteins are administered 48h post-TBI; (2) determine whether all 3 proteins or only a subset of the proteins are required for increasing 30-d survival when dosed 48h post-TBI; (3) determine whether the 3 drug combination (+/- ACEI) can improve 30-d survival when administered at even later times post-TBI, e.g., 72h; and (4) perform a pharmacokinetic and mechanistic study of the 3 proteins and the ACEI in TBI mice to measure effective plasma levels of the 3 proteins and the ACEI over time, and to determine if increased 30d survival correlates with a more rapid increase in hematopoietic progenitor cell numbers early after irradiation in the mice. Additional mechanistic studies include performing complete blood cell analyses at various times post-TBI to determine if increased 30-d survival correlates with accelerated recovery of neutrophils, platelets, and other peripheral blood cell types. In addition, we will finalize GMP manufacturing processes and identify stable liquid formulations of the 3 proteins for use in future IND-enabling studies. These studies will lead to the development of a life-saving treatment option for a heretofore untreatable H-ARS population ? patients who cannot be treated until 48h or later post-TBI.